Nanopump devices and methods

ABSTRACT

Disclosed is a nanopump for pumping small volumes of electrolyte solution under the control of a voltage source. The device includes a chamber and a nanopore membrane which partitions the chamber into upstream and downstream regions. When a voltage potential is applied across the membrane, electroosmotic flow through the membrane channels produces a precise-volume flow between the two chamber regions. Also disclosed is a method for precis-volume pumping employing the nanopump. Also disclosed is device for determining the lengths of nucleic acid fragments in an electrolyte solution of different-length fragments is disclosed. The device includes a chamber having disposed therein, a nanopore channel extending between upstream and downstream chamber regions. By applying a voltage potential across upstream and downstream electrodes in the chamber regions, individual nucleic acid fragments contained in the solution are moved through the channel. A current detector detects time-dependent current flow across said membrane, and from the measured flow times, fragments lengths can be estimated. Also disclosed is a device for separating macromolecules in a solution of macromolecules having different molecular sizes. The device includes a separation chamber having upstream and downstream ends, and one or more nanopore membranes disposed in the chamber between said upstream and downstream ends, partitioning the chamber into two or more chamber regions, respectively. Upstream and downstream electrodes are disposed at the upstream and downstream ends of the chamber, respectively. A controller in the device has a power source operatively connected to the electrodes for applying a selected voltage potential across said channel, to pump solution through each of said membranes, in an upstream-to-downstream direction, wherein macromolecules contained in the solution are filtered at each successive membrane, to concentrate successively smaller macromolecules in successively more downstream chamber regions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. ProvisionalPatent Application Serial No. 60/298,830 entitled “Nanopump Apparatusand Method” filed on Jun. 15, 2001; U.S. Provisional Patent ApplicationSerial No. 60/298,812 entitled “Nanopump Separation Device and Method”filed on Jun. 15, 2001; and U.S. Provisional Patent Application SerialNo. 60/298,813 entitled Nanopump Device for Determining DNA FragmentLengths” filed on Jun. 15, 2001, the disclosures of which areincorporated as if fully rewritten herein.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of (i) small-volumepumps, applications thereof, and methods employing such pumps; (ii)separation devices, and methods using such devices; and (iii) devicesand methods for determining DNA fragment lengths, e.g., for DNAsequencing.

SUMMARY OF THE INVENTION

[0003] A. Nanopump Apparatus and Method

[0004] The invention includes a nanopump for pumping electrolyte fromone reservoir to another, or from one reservoir to an electrolyte“sink.” The pump includes a supply reservoir for holding a supplyelectrolyte solution, and disposed against a wall region of thereservoir, a membrane having a plurality of flow-through channelsextending between an inner membrane surface in contact with said supplyelectrolyte solution, and an outer side in contact with the electrolytesink. The channels have a minimum cross-sectional dimension between 2and 100 nm and a net surface charge when the pH of the supply solutionis within a given pH range. Electrodes are disposed on either side ofmembrane for contact with said supply and recipient solutions. Acontroller includes a power source operatively connected to theelectrodes for applying a selected voltage potential across saidchannel, to pump solution in the supply reservoir through said channel.

[0005] The channels forming the membrane preferably have a substantiallyuniform minimum dimension along their lengths, and more preferably aminimum dimension in a selected range between about 10 and 30 nm.

[0006] The controller is preferably operable to apply a voltagepotential across the electrodes of between about 0.5-20 volts. Thecontroller may additionally include a control element for applyingacross the electrodes, a pulsed voltage whose duration is effective topump a selected volume of supply solution across said membrane.

[0007] The membrane may be formed of a silicon substrate withpolysilicon and silicon dioxide layers, or other suitable materials suchas polymers, titanium, etc. The pump may include a recipient reservoirdesigned for holding the recipient solution in contact with the outerside of said membrane.

[0008] Also forming part of the invention is a method of pumpingcontrolled and reproducible nanoliter (nl) quantities of an electrolytesolution from a supply reservoir into a recipient electrolyte solution.The method includes placing the electrolyte solution in a supplyreservoir, in contact with a membrane of the type described above.placing the outer membrane surface in contact with a recipientelectrolyte solution, and with a pair of electrodes placed across saidmembrane, in contact with solution in said supply reservoir and withsaid recipient electrolyte, applying across the electrodes, a voltagepotential effective to pump supply solution across the membrane from thesupply reservoir into the recipient solution via electroosmotic flow.

[0009] B. Nanopump Separation Device and Method

[0010] The invention includes a separation device for separatingmacromolecules in a solution of macromolecules having differentmolecular sizes. The device includes a separation chamber havingupstream and downstream ends, and one or more membranes disposed in thechamber between the upstream and downstream ends, partitioning saidchamber into two or more chamber regions, respectively. Each membranehas a plurality of flow-through channels extending between adjacentchamber regions, the channels have a selected minimum cross-sectionaldimension in the range between 2 and 100 nm and a net surface chargewhen exposed to a solution within a given pH range, and if the devicecontains two or more such membranes, the selected minimum cross-sectionof the channels in any membrane is greater than that in membraneimmediately adjacent in the downstream direction,

[0011] Upstream and downstream electrodes are disposed in the chamber,for contacting solution placed in the chamber and contained in the inthe upstream-most and downstream-most of the chamber regions,respectively. A controller in the device includes a power sourceoperatively connected to said electrodes for applying a selected voltagepotential across the membrane(s), to pump solution through each of themembranes, in an upstream-to-downstream direction, such thatmacromolecules contained in the solution are filtered at each successivemembrane, to concentrate successively smaller macromolecules insuccessively more downstream chamber regions.

[0012] The device preferably includes at least two membranes, and atleast three separate chamber regions. For separating globular proteinshaving solution radii between about 20 to 300 angstroms.

[0013] The device may further include a fluid outlet communicating witheach chamber region, and an electrode associated with each outlet. Herethe voltage source is operably connected to the outlet electrodes, fordiverting macromolecules in a chamber region out of the chamber throughsaid outlet.

[0014] Alternatively, the each chamber region has inlet and outlet portsfor circulating liquid through that chamber, to remove separatedmacromolecules in the chamber region from the chamber,

[0015] Also forming part of this invention is a method of separatingmacromolecules in a solution of macromolecules having differentmolecular sizes. The method includes placing the electrolyte solution inan upstream chamber region of a device of the type described above, andwith a pair of electrodes placed across the membrane(s), in contact withsolution in the upstream and downstream reservoirs, applying across saidelectrodes, a voltage potential effective to pump supply solution acrosssaid membrane from the upstream into the downstream reservoir, whereinmacromolecules in said solution are separated on the basis of theirability to pass through the channels in said membrane.

[0016] C. Nanopump Device for Determining DNA Fragment Lengths

[0017] The invention includes a device for determining the lengths ofnucleic acid fragments in an electrolyte solution of different-lengthfragments and having a selected pH. The device includes a chamber, and amembrane disposed in said chamber and having a channel extending betweenan upstream chamber region adapted to hold the electrolyte solution ofsuch different-length fragments, and a downstream chamber region adaptedto hold an electrolyte solution, where (i) said channel has a selectedminimum cross-sectional dimension in the range between 2 and 15 nm and anet surface charge within a given pH range that includes the selectedsolution pH. Upstream and downstream electrodes disposed in the upstreamand downstream chamber regions, respectively, contact solution placed inthe corresponding chamber regions. A controller in the device includes(i) a power source operatively connected to the electrodes for applyinga selected voltage potential across the channel, to move individualnucleic acid fragments contained in the solution through the channel,and (ii) a current detector for detecting time-dependent current flowacross the membrane.

[0018] The channel may be formed by a pair of planar channel, eachhaving a channel thickness in the 2-20 nm range, arranged orthogonallywith respect to one another, such that the intersection of the twochannels a forms a rectangular area whose dimensions correspond to thetwo thicknesses of the intersecting channels.

[0019] The membrane may have a plurality of channels, where the currentdetector is effective to measure time-dependent current independentlythrough each of the channels. The minimum channel width is preferablywithin the range 2-10 nm.

[0020] The controller may be effective to place across the electrodes, avoltage potential effective to move nucleic acid fragmentselectrophoretically through said channel.

[0021] Also disclosed is a method of determining the lengths of nucleicacid fragments in an electrolyte solution of different-length fragmentsand having a selected pH. The method includes placing the solution in anupstream chamber region of a chamber having upstream and downstreamchamber regions separated by a membrane having a channel extendingbetween the two chamber regions, where the channel has a selectedminimum cross-sectional dimension in the range between 2 and 15 nm and anet surface charge within a given pH range that includes the selectedsolution pH. With a pair of electrodes placed across said membrane, incontact with solution in the upstream and downstream chamber regions, avoltage potential effective to pump solution across said channel,wherein individual nucleic acid fragments move through said channel isapplied across the membrane. Time-dependent changes in current flowthrough the channel are determined, as a measure of the length ofindividual nucleic acid fragments moving through said channel.

[0022] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 illustrates the pH dependence of charge on a siliconsurface;

[0024]FIG. 2 illustrates the pH dependence of charge on a siliconsurface derivatized with amine groups;

[0025]FIG. 3 illustrates the principle of electro-osmotic flow in achannel having charged surface groups;

[0026]FIG. 4 shows, in simplified view, a nanopump constructed inaccordance with the invention;

[0027]FIG. 5 shows plots of flow rates in the nanopump of the inventionas a function of applied voltage, for various pore sizes;

[0028]FIG. 6 illustrates electroosmotic flow principles in a nanoporepump;

[0029] FIGS. 7A-7D illustrate steps in the production of a nanoporefilter used in the nanopump of the invention;

[0030]FIGS. 8A and 8B illustrate the operation of a nanopump filtrationdevice constructed in accordance with the invention;

[0031]FIGS. 9A and 9B illustrate the operation of a nanopumpdrug-delivery device constructed in accordance with the invention;

[0032]FIGS. 10A and 10B illustrate the construction of a nanoporeopening formed by two nanopore planar channels;

[0033]FIG. 10C shows key elements in a nanopore device constructed inaccordance with the invention for determining DNA fragment lengths;

[0034]FIG. 11 illustrates elements of a DNA sequence detecting deviceconstructed in accordance with the invention; and

[0035]FIG. 12 illustrates electrokinetic forces acting on a DNAmolecules bound to a wall surface in the SNP-sequence detecting device.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention, called a “nanopump”, is a fluid pumpcomprising a collection of parallel arrays of multiple channels which,in their smallest dimensions are in the approximate range of 5 nm-100nm. The channels, e.g., rectangular or U-shaped channels, are fabricatedfrom materials such as silicon and silicon oxides, polymers, or metals,such as titanium, the surfaces of which exhibit a net surface chargewhen exposed to electrolyte solutions at appropriate pH levels. Underthe influence of an electrical field applied across the array,electroosmotic flow is induced, pumping aqueous subject matter from oneside of the array to the other.

[0037] By controlling the geometry of the multiple-channel arrays, theindividual channel width, surface charge density and polarity, togetherwith controlling the array thickness and electrolyte properties, theflow properties can be tailored for highly specific applications.

[0038] A. Theory of Operation

[0039] The nanopump is based on electroosmotic flow. Electroosmotic flowis a basic physical-chemical phenomenon, but has special properties whenit is applied to materials such as silicon or silicon oxides. A furtherexamination of silicon (Si) surface chemistry is required to ensure thatthe basic process is well described.

[0040] The chemical state of the silicon surface can be either in anoxidized form or oxide-free, bare silicon, terminated by Si—H groups.The silicon surface, after a wet cleaning step and a final oxidativestep, is hydrophilic. The thickness of this SiO₂ layer is between 0.6and 2.0 nm, its nominal value depending on oxidation conditions and themeasurement techniques used. This chemical oxide, often referred to as“native” oxide, forms a passivation layer with a dangling-bond defectdensity in the range of 10¹² cm⁻² at the Si/SiO₂ interface. The defectdensities reported are about two orders of magnitude higher than forthermal oxides. Recent studies suggest that the quality of the nativeoxides is strongly dependent on the applied chemistry in which they areformed, and that higher-quality oxides can be obtained with alternatechemistries such as DI water/ozone.

[0041] Wet-chemically grown oxides are hydrophilic in contrast tothermal oxides. This difference is caused by the way the oxygen atom isbound to the silicon on the surface. Thermal oxides are characterizedthrough the formation of siloxane rings, which are very stable againsthydrolysis. Wet-chemically grown oxides are generally covered withsurface hydroxyl groups (Si—OH) called silanol groups, and are verysimilar in their behavior with respect to silica gels. Geometricalconsiderations and chemical measurements indicate an average surfacedensity of around five hydroxyl groups per nm², but this number cantypically range from 2-12 hydroxyl groups per nm². It is important tonote that not all hydroxyl groups formed on a surface are chemicallyequivalent owing to structural differences in their coordination, but ingeneral the surface hydroxyl groups on a hydrous oxide have donorproperties similar to those of their corresponding counterparts insolution, the hydroxides.

[0042]FIG. 1 illustrates the dissociation of silanol(−) groups, such asgroups 16, at the surface 18 of a silicon substrate 20, at low and highpH extremes. The adsorption of metal ions and protons can be understoodas competitive complex formation with deprotonated surface groups(Si—O—) which behave like Lewis bases. This means that the adsorption ofspecies on a hydrous oxide surface of Si can be compared with complexformation reactions in solution. However, the extent of adsorption alsodepends strongly on the surface charge of the oxide i.e., the number ofhydroxyl groups and the degree of dissociation and on the pH of thesolution. Silanol groups are completely ionized at pH levels above 9(right in FIG. 1), creating a negative surface potential. Below pH 4(left in FIG. 1) the silanol groups are protonated and the surface 20 isvirtually neutral. Between these two pH extremes, the surface becomesprogressively more deprotonated, and negatively charged, with increasingpH.

[0043]FIG. 2 shows a silicon substrate having a portion of its silylgroups are modified to amino groups, such as groups 24. Methods forderivatizing a glass or silicon surface with amino groups are wellknown. A hydrolytically stable amino-silica glass coating material canbe applied on the inner surfaces of nanopump channels in order to createa surface which, depending on the pH of the medium, can be eitherpositively or negatively charged. Aminopropyltriethoxysilane, or asimilar silane reagent, is used to introduce primary amino groups ontothe silica surfaces.

[0044] Following such treatment, as illustrated in FIG. 2, the netcharge at the surface of the coating material depends on the degree ofprotonation of the amino groups and the degree of ionization of thesilanol groups, thus enabling manipulation of the magnitude anddirection of the electroosmotic flow (EOF). At lower pH (at levelssomewhat below pH factor 6.0), the coating bears a net positive charge,which results in an electroosmotic flow from the cathode toward theanode and minimizes the wall-solute interactions of basic species. Athigher pH (at levels somewhat above pH factor 6.5), the coating surfacebears a net negative charge and the coated nanopore behaves like anuncoated one, having an EOF in the cathodic direction. Such anamino-silica glass coating is extremely stable under both acidic andbasic conditions.

[0045] Electroosmotic flow is also influenced by addition of certainorganic bases to a running buffer. For example, addition ofN,N,N′,N′-tetramethyl-1,3-butanediamine (TMBD) in the runningelectrolyte effects electroosmotic flow and the migration behavior ofbasic proteins in bare fused-silica capillaries. Depending on theelectrolyte pH (4.0, 5.5 and 6.5, respectively) and additiveconcentration the electroosmotic flow can be either cathodic or anodic.A similar Langmuirian-type dependence of the electroosmotic flow on theconcentration of TMBD in the running electrolyte was found at the threeexperimented pH values, which may be indicative of the specificadsorption of the additive in the immobilized region of the electricdouble layer at the interface between the capillary wall and theelectrolyte solution.

[0046] B. Electroosmotic Flow

[0047] Most surfaces, including silicon as noted above, obtain a surfaceelectric charge when they are brought into contact with electrolytesolutions. This surface charge influences the ion distribution in thepolar medium forming the electric double layer. Gouy and Chapman modeledthe region near the surface as a diffuse electrical double layer (EDL),where they equated the non-uniform ion distribution to the competingelectrical and thermal diffusion forces. Stern later presented the basisfor the current model, in which the Stern plane splits the EDL into aninner, compact layer and an outer, diffuse layer.

[0048] In the inner layer, also known as the Stern layer, the geometryof the ions and molecules strongly influences the charge and potentialdistribution, with the Stern plane located near the surface at roughlythe radius of a hydrated ion. The inner layer between the surface andthe Stern plane is considered to be immobile. When the ions are withinthe Stern plane, thermal diffusion will not be strong enough to overcomeelectrostatic, or Van der Waals forces and they will attach to thesurface to become specifically adsorbed.

[0049] In the outer diffuse layer, the ions are far enough away from thesurface that they are mobile. Electrokinetic transport phenomena such aselectroosmosis can be understood in terms of the surface potential atthe surface of the shear (approximately at the Stern plane), known asthe zeta potential (ξ), because these phenomena are only directlyrelated to the mobile part of the EDL.

[0050] Because of the EDL, the net charge density (σ_(e)) within thediffuse layer is not zero. If an electric field is applied along thelength of the channel, a body force is exerted on the ions in thediffuse layer of the EDL. The ions will move under the influence of theapplied electrical field, pulling the liquid with them and resulting inelectroosmotic flow. The fluid movement is carried through to the restof the fluid in the channel by viscous forces. This electrokineticprocess is called electroosmosis.

[0051]FIG. 3 represents anodal electroosmotic flow induced through achannel 26 filled with an electrolyte solution 28. The channel is linedwith negatively charged ionized silanol groups, such as groups 30.Potential difference is established by placing electrodes 32, 34 atopposite ends of channel. Since fluid motion is initiated by theelectrical body force acting on the ions in the diffuse layer of theEDL, electroosmotic flow depends not only one the applied electricalfield but also the net local charge density in the liquid.

[0052] Prior studies of EDL and electroosmotic flows are limited tosystems with simple geometries such as cylindrical capillaries withcircular cross sections and slit-type channels formed by two parallelplates. However, for the channels embodied in the present invention, andin other fluidic devices produced by micro-machining techniques, thecross-sectional shape is close to planar. In such a situation, the EDLfield is two-dimensional and will influence the two-dimensional flowfield in the rectangular microchannel.

[0053] B. Pump Structure

[0054]FIG. 4 is a pictorial representation of a nanopump 38 constructedaccording to the present invention. The pump includes a nanopore filteror membrane 40 providing a plurality of rectangular-shaped(substantially planar) parallel nanochannels, such as microchannels 42,each channel connecting a donor or supply reservoir 44 to receiver orrecipient reservoir 46. The nanopump is fabricated from silicon-basedmaterials using the techniques described herein. A plurality of ports 48feed donor reservoir 44 and a second plurality of ports 50 draw excessfrom the receiver reservoir 46. A pair of electrodes 52, 54 are used toapply a potential difference across the membrane are suitably connectedto a power source.

[0055]FIG. 5 is a graph that depicts the relationship betweenelectrolyte flow rate and voltage applied to nanopumps with differentchannel widths. In the preferred embodiment, a nanopore membrane isplaced between two chambers. This structure promotes electroosmotic flowwhen a potential difference is applied across the membrane. Theresultant flow rate is related to the porosity of the membranes and tothe size of the channel. Unexpectedly, optimal flow rates are observedat a channel dimension of about 20 nm. Greater channel dimensions, e.g.,27 and 49 nm, exhibit lower flow rates. The data in FIG. 5 were obtainedusing channels with poly-silicon and crystalline silicon materialsidewalls with a pore geometry that is about 45 μm in length, about 20to 40 nm in width, and about 5 μm in depth.

[0056]FIG. 6 illustrates the relationship between channel width and flowrate. This phenomenon may be partially related to the increasedproportion of the overall channel volume occupied by the diffuse ionicdouble layer as the channel width is decreased. Flow rate is alsoinfluenced by electrolyte properties including pH, ionic strength andionic species.

[0057] In operation, the reservoirs in the pump are filled with anelectrolyte solution whose pH is effective to impart a net charge, e.g.,net negative charge, to the walls of the nanopore membrane. The powersource is then activated to apply a selected voltage potential acrossthe electrodes, producing electro-osmotic pumping across the membranefrom the supply to the recipient reservoir.

[0058] C. Fabrication Process

[0059] The nanopump is created through a microfabrication process usingbulk and surface micromachining. In the preferred embodiment, themicrofabricated nanopump comprises a surface-micromachined array ofchannels on top of an anisotropically etched silicon wafer that providesmechanical support. The selection of channel pore size (the minimumchannel dimension) is in 5-100 nm range, e.g., 5, 10, 20, 30, 50 or 80nm, preferably 10-30 nm pore size.

[0060] To reach a desired pore size in the tens of nanometers range,strategies have been developed based on the use of a sacrificial oxidelayer sandwiched between two structural layers. This process isdiscussed in the co-pending patent application incorporated herein byreference. Because the flow rate of a nanopump varies according to thematerial being pumped, it is important to tailor new nanopumps forspecific applications as discussed above.

[0061] A nano-channel is formed by sandwiching a SiO₂ sacrificial layer,the thickness of which determines the nominal pore size, between apolysilicon structural layer and the silicon wafer. For this design,increasing the number of entry holes maximizes the flux. Phosphatebuffered saline (PBS) fluxes as high as 1.0 mL/cm2-hr have been attainedfor membrane filters with 30 nm-sized pores. Filtration tests showedgreater than 99% of 100 manometer beads (actual log reduction of greaterthan 5) were retained with 50 nanometer pores.

[0062] FIGS. 7A-7D are pictorial representations of the cross-section ofa wafer after successive stages in the microfabrication process used tocreate nano-channel arrays. The nano-channel arrays are microfabricatedfrom silicon and silicon nitride (Six,Ny) architectures. The criticaldimension (i.e., width) of the nanopump channels will be defined by thethickness of sacrificial silicon oxide films 60, a parameter that can becontrolled to sub-nanometer resolution. The fabrication process allowsfor dense arrays of nano-channels, thus improving the utility of thesearrays for pumping applications. The fabrication process is summarizedhere for review. A detailed and specific discussion of this fabricationprocess is presented, for example, in U.S. Pat. Nos. 6,044,981,5,985,328, 5,985,164, 5,948,255, 5,928,923, 5,798,042, 5,770,076, and5,651,900, all of which are incorporated herein by reference.

[0063] As a first step, and with reference to FIG. 7A, a structuralpolysilicon layer 56 is deposited over a silicon nitride layer 58 (anetch-stop layer) and etched with the nanopore mask. Following this, asacrificial oxide layer 60 (FIG. 7B) is grown using thermal oxidation todefine the nanopump array thickness. Thermal oxidation gives thicknesscontrol to sub-nm resolution across in entire 4″ silicon wafer.

[0064] A second polysilicon structural layer 62 is then deposited overthe pores and planarized to allow access to the nanopump channels fromthe front face of the array. A silicon nitride protective layer is thendeposited and etch holes, such as hole 64 are opened on the backside ofthe wafer. The bulk silicon is removed through these etch holes up tothe etch stop layer of the array, giving the structure shown in FIG. 7C.

[0065] The structure is then released by etching the protective nitridelayers and the sacrificial oxide layers in a concentrated HF bath. Inparticular, sacrificial layer 60 is etched in the region between entryholes and the lower opening 64, producing defined-size nanopore channels68 (FIG. 7D). Surface modification of the polysilicon structure revertsit to a hydrophilic surface, thus making it useful as a filter forbio-fluids.

[0066] D. Molecular Filtration and Separation

[0067] Many clinical and research applications involve separation ofspecific biological molecules, such as nucleic acids and proteins, forfurther analysis and characterization.

[0068]FIGS. 8A and 8B show the basic elements of a nanopump basedseparator device 70 constructed in accordance with the invention. Thedevice generally includes a separation chamber 70 having upstream anddownstream ends 72, 74, respectively, and a plurality of membranes 76,78, 80 that partition the chamber into a plurality of separation regions82, 84, 86, and 88, including an upstream chamber region 82 and adownstream chamber region 88. Each membrane, such as membrane 76, is ananopore filter of the type described above, and has a selected channelsize for filtering macromolecules, e.g., proteins or nucleic acidfragments, of selected sizes.

[0069] The device also includes electrodes 90, 92 disposed in theupstream and downstream chamber regions, respectively, and a powersource 94 connecting the two electrodes. Each chamber region may furtherinclude a side inlet and outlet (not shown) for removing liquidcontained in each chamber region.

[0070] In operation, a mixture of macromolecules or other solute speciesand, optionally, small particles, such as virus particles or colloids orliposomes, in an electrolyte are placed in the downstream chamberregions, and the other chamber regions are filled with a suitableelectrolyte solution, in particular, one whose pH supports a net chargeon the membrane wall surfaces. By applying a selected voltage across theelectrodes, liquid in the each chamber is pumped in a downstreamdirection across the immediately downstream membrane. At each membrane,macromolecules that are larger than the membrane pore size will beretained in the upstream channel region, and those that are smaller willpass through into the downstream channel region.

[0071]FIG. 8A shows the device with a sample 96 of different-sizedmacromolecules in an electrolyte solution placed in upstream channelregion 82, and a suitable electrolyte solution placed in each of theupstream channel regions. When a voltage of appropriate polarity andfield strength is applied across electrodes 90, 92, sample 96 is drawnfirst through membrane 76, which discriminates between the largestmacromolecules (or particles) in the sample and all other samplespecies. Similarly, as solution in the next upstream channel region 84migrates is pumped through membrane 78, this membrane blocks passage ofmacromolecules of a certain size, and passes others, producing asuccessively greater concentration of smaller-sized macromolecules ateach upstream channel region. At the end of the filtration process, themacromolecules contained in each channel region, including theupstream-most region, can be removed, e.g., by flowing a lateral streamof electrolyte through each channel region.

[0072] The system of stacked nanopumps described above may be modifiedfor performing the specific isolation and purification of a particularprotein or oligonucleotide from a complex mixture or crude cell extractrapidly and precisely using electroosomotic flow. For specific proteinseparation, the surfaces of the silicon nanopump channels can bechemically modified to contain covalently linked ligands with strong andspecific affinity to their conjugate. The chemistry of silanization iswell established and can be easily modified for specific ligandattachment for use to capture a variety of enzyme families.

[0073] For specific oligonucleotide isolation and purification, separatenucleic acid oligonucleotides complementary to the nucleic acid sequenceintended for capture can be synthesized relatively inexpensively usingtraditional synthesis methods. These capture oligonucleotides could thenbe covalently linked to the surfaces of the nanopump channels usingestablished methodology and can be used to capture any specificoligonucleotide of interest.

[0074] Most importantly, this device allows for the simple and rapidpurification of multiple specific biomolecules at the same time. Eachnanopump level can be ‘programmed’ to collect a specific molecule in acomplex mixture by simply by inserting the correct nanopump ‘module’primed with the appropriate protein capture ligand or nucleic acidoligonucleotide. In simple terms, this device is capable of performingnumerous molecular isolations from one sample with one simpleelectroosmotic run that is both user-customizable and scalable.

[0075] In summary, the proposed device has several advantages overcurrent laboratory methods for rapid and precise separation,purification and isolation. With the use of nanopump array modules,exact size-exclusion fractionation can be performed on multiple complexsamples in less time and with precise resolution. Also, specificpurification and isolation for a variety of molecules can be recoveryefficiency. Finally, this device can perform the separation, filtration,and affinity purification of biomolecules by simply switchinguser-customizable modules, eliminating the need for multiple expensiveelectrophoretic and liquid chromatographic equipment.

[0076] E. Implantable Drug-Delivery Device

[0077]FIGS. 9A and 9B illustrate an implantable drug delivery device 100constructed according to the present invention, and shown at initial andhalf-spent stages of operation, respectively. The device generallyincludes a housing 102 having a delivery port 104. The housing is formedof a suitable biocompatable material that allows its placement at animplantation site in a body.

[0078] Contained in the housing is a supply reservoir 108, a nanopump110, constructed in accordance with the invention, and a one-way checkvalve 112 that allows one-way flow of solution contained in reservoir108 from the pump to the exit port, via a channel 103. The reservoir,pump and valve are all part of a continuous flow system, as shown. Alsoshown is a vent structure 114 that includes a vent 116 and a plunger 118disposed within reservoir 108. As solution is pumped out of thereservoir through exit port 104, the reservoir space is progressivelyfilled by movement of plunger 118 toward the pump, as indicated in FIG.9B, with the reservoir volume upstream of the plunger being filledprogressively by fluid available at the implantation site.

[0079] The nanopump is activated by means of a pair of electrodes 120,122 disposed on either side of a nanopore membrane 125 in the pump, andconstructed in accordance with the invention. More particularly,electrode 122 is contained within an upstream supply reservoir 124 inthe pump, and electrode 120, within a downstream recipient reservoir 126in the pump. Application of a voltage potential across the twoelectrodes is effective to pump electrolyte drug solution in adownstream direction, in accordance with the invention.

[0080] The power source in the device, for applying a potential acrossthe pump electrodes, is a battery pack indicated at 130. The batterypack has a voltage of preferably 1-5 volts. Activation of voltage fromthe battery pack to the electrodes is through a timing control unit 132which is a small processor designed to produce an activation signal fora selected signal duration, e.g., 1-5 seconds, at selected timeintervals, e.g., every 4 or 8 hours.

[0081] In operation, the device, including the supply reservoir and thenanopump, is filled with an electrolyte solution, typically a drugsolution having a selected drug concentration. Before implantation, thecontrol unit is adjusted to produce desired metered amounts of drugsolution, e.g., pl to nl amounts, at selected time intervals, e.g.,every eight hours. The loaded, programmed device is then implanted at aninternal body site, e.g., at a subcutaneous site, to deliver a meteredamount of drug solution at selected time intervals.

[0082] Power requirements to induce electroosmotic flow are to be lowdue to the relatively porosity provided by the multiple parallel channelarrays. Moreover, the same geometry favors fluid flow. Exquisite on-offcontrol, and thus control of drug input rate and pattern, is provided bythe selected switching circuitry. For example, pulse, square wave andcontinuous patterns are achievable simply by controlling the length oftime the switch 127 is closed. As described, having a miniaturized timerin the circuitry provides a means of providing “on-board” intelligence.Alternatively, the switch may be closed magnetically. Thus externalcontrol is possible by simply placing a magnet close to the skinsurface, providing a drug input rate and pattern controlled by thepatient himself, or his caregiver.

[0083] F. Sizing Nucleic Acid Fragments

[0084] The nanopump described herein can be adapted, in accordance withanother aspect of the invention, to perform oligonucleotide lengthanalysis using a rapid, one-step process that potentially limits complexsample preparation. For this application, a silicon micromachinedmembrane that contains an array of nano-sized pores with dimensions ofapproximately 2 nm is fabricated. An electrical potential across thepore is used to assist in moving oligonucleotides from the samplethrough the pores. The pore geometry is selected to allow singleoligonucleotides to proceed through the pore in a linear fashion (i.e.end-to-end).

[0085] A separate sensing array allows for monitoring the pore blockageby nucleic acid molecules as a function of time. For example, examiningthe change in electrical potential across the pore as theoligonucleotide moves through it can provide a measure of fragmentlength (i.e. number of base pairs). It is common in the study ofneurobiology and biophysics to form biological membranes or planar lipidmembranes (such as lipid bilayer films) and to introduce nano-sizedchannels. These channels can be made to stay open for extended periodsof time. Kasianowicz et. Al, Proc Nat Acad Sci (USA), 93(24):13770(1996) reasoned that applying a transmembrane voltage could causepolyanionic oligonucleotides to flow through a membrane channel as anextended linear chain. When these molecules were present in thechannels, they could be detected as a reduced or blocked level of normalionic flow measured as reduced ionic current. Measurement of the timeand magnitude of such blockages was demonstrated as a method ofrecording the molecular length, and other characters, of the particularmolecule.

[0086] In the case of the nanopump envisioned here, the biologicalmembrane is replaced with a membrane containing nanopores that arefabricated using silicon micromachining, as described above, but wheretwo “planar” channels placed end to end and at right angles with respectto each other, to form a one-dimensional (1D), pore (i.e. channel)shaped like a rectangular parallelepiped. The pore geometry can bedesigned with precise and selectable nano-sized channel widths in the2-50 nm size range. The pore height and pore length can also be designedwith microsized dimensions in the 1-50 nm size range.

[0087] A diagram of a typical 2D nanopore channel 128 is shown in FIG.10A. A selected number of pores, with the typical shape shown in, arenormally fabricated into a planar, membrane-like structure, as describedabove.

[0088] In order to determine strand length the DNA strand must traversea man-made nano-sized pore described above in a linear fashion. This isaccomplished, in accordance with one embodiment of the invention, byusing a second 2D pore 130 located adjacent and perpendicular to thefirst 2D pore as seen in FIG. 10B. Using this design, the intersection132 between the microfabricated pores can have nano-size dimensions inboth planes and a nano-sized pore-length so it more closely approximatesa 1D (that is, linear as opposed to planar) biological pore.

[0089] Native DNA consists of one long molecule that makes up achromosome. It is a two-stranded spiral (double-helix) that includesabout 3 billion nucleotides arranged in subunits called base pairs. Asegment of DNA carrying genetic instructions, or gene, is approximately100,000 base pairs long. To perform many DNA analytical experimentation(e.g. sequencing, Southern hybridization) the long DNA molecule isusually digested into smaller, single-stranded fragments that typicallycontain <500 base pairs (FIG. 12). The diameter, D, of asingle-stranded, 100 base pair oligonucleotide is approximately 2 nm andthe length, L, is approximately 200 nm. In the present invention, bymeasuring the exact time necessary for the passage of one moleculethrough the channel, it is possible to correlating this time with thelength of the molecule. In this manner, exact oligonucleotide length canbe established.

[0090] The construction of an electronic device 131 for measuringfragment length, based on the above principles, is shown FIG. 10C. Inthis case, a silicon micromachined, 1D nano-sized pore is created asdiscussed above, at the intersection of two channel-forming substrates128, 130. The size of this pore, shown at 132, is S_(p)×S_(p), measuring2 nm×2 nm. The pore's channel length, L_(p), is selectable within arange of 5 to 50 nm.

[0091] Electrodes 134 (E1), 136 (E2) located in buffer solutions can beplaced on both sides of the nanopore membranes or attached to thesurfaces of the silicon membrane at a point near the pore location, asshown in the figure. When the device includes an array of such pores, anarray of surface-attached electrodes can used to monitor the ion currentpassing through more than one pore. Surface metalization of silicondoping methods, common to the microelectronic industry, can be used toform single electrodes or electrode arrays on the surface of thesilicon. That is, multiple pores can be created and each pore can bemonitored by a separate set of E1 and E2 electrodes. Another option tomultiple-pore monitoring is to have a common E1 electrode and an arrayof E2 electrodes. Using multiple pores will allow for a more rapid dataacquisition rate and a more extensive analysis.

[0092] Another advantage to a microfabricated nano-pore membrane channelis that a third electrode 138 (E3) can be added to the channelarchitecture, providing the advantage of using a fixed voltage of commonE1 and E2 electrodes for pumping through the channel, while monitoringion current using an array of E3 electrodes. The E3 electrode must be onthe order of 5 nm thickness, and may be created by doping the siliconitself to increase its conductivity using methods common in themicroelectronic industry.

[0093] G. DNA Sequence Analysis

[0094] A basic design of a nanopump array intended for DNA sequenceanalysis, in accordance with another aspect of the invention, is givenin FIG. 11. The central element 140 includes a series of nanopumpchannel arrays, such as arrays 142, 144, micromachined out of a singlesilicon wafer.

[0095] The nanopump array layer is sandwiched between upper and lowermicrofabricated layers 146, 148, respectively, upper layer 146 is fittedwith discrete “donor” reservoirs, such as reservoirs 150, 152, whichalign above and are continuous with one side of the individualnanopumps. Each of the upper donor reservoirs is fitted with anelectrode, such as electrodes 154, 156 in reservoirs 150, 152,respectively. A similar reservoir layer 148 is aligned below thenanopump layer, but in the case of this lower layer, the individualreceiving reservoirs, such as reservoirs 158, 160, are also connectedvia channels, such as channel 162, 164, to a “common” receivingreservoir 166.

[0096] An optical flow-cell 168 is included in the channel leading tothe receiving reservoir and a fluorescence optical detection system ispositioned to measure the fluorescence signal of the fluid flowingthrough this common channel. Each of the three layers is aligned andsealed so that each nanopump is continuous with the corresponding donorand receiving reservoirs. The reservoirs and connecting channels arefiled (primed) with electrolyte solution prior to use.

[0097] After fabrication and assembly, the surfaces of the channels arechemically grafted with reactive chemical groups (such as primary aminoor thiol groups) using standard silane reagents. Alternatively thechannels may be linked with a ligand such as avidin (or its bindingpartner/receptor, biotin). Capture DNA sequences are subsequentlygrafted to the surface directly or through avidin/biotin interactions.For attachment, the capture sequences are added to the common receivingreservoir and a voltage difference is applied between the commonreservoir (anode) 167 and any or all of the individual donor reservoirs(cathodes). The DNA sequences migrate under the influence of theelectric field from the common reservoir through the chemically modified(or ligand modified) nanopump channels, into the donor reservoirs. Thepolarity may be periodically reversed (cycled) to optimize the chemicalgrafting of the capture DNA to the nanopump channel surfaces as theypass. After capture DNA sequence grafting, the reservoirs and channelsare washed by passage of buffer throughout the system.

[0098] Samples of fluorescent-labeled DNA probes of known sequence areadded to the individual donor (upper) reservoirs. Current is passedthrough all the nanopumps in the array (anode in donor reservoirs,cathode in receiving reservoirs). As the probe DNA sequences passthrough the nanopump channels, hybridization occurs. Again, cycling ofthe electrode polarity between the donor and receiving reservoir, andthus reversing the electrophoretic movement of the DNA probes, may beused as a strategy to promote optimal hybridization to capture DNAsequences bound to the nanopump channel surfaces.

[0099] Dehybridization of probe and capture DNA sequences is controlledby a combination of applied current (electrophoretic force, EF) andelectroosmotic flow (tangential fluid flow force, TFFF). Importantly,these two forces may be in the same or opposite directions and may beapplied as a gradient or pulsed. For example in the case of negativelycharged DNA molecules (DNA is highly negatively charged by virtue ofcharged phosphate groups in each residue of the backbone structure), EFwill always be in the direction of the cathode. Electroosmotic flow, andhence the TFFF, may be either cathodal or anodal, depending on the netsurface potential at the plane of fixed charges on the nanoporesurfaces. By adjusting and fine tuning EF and TFFF, the selectivity ofdenaturation of hybridized DNA will be improved, thus allowing for thediscrimination of dehybridization of probe and capture DNA with fewermismatched base pairs. SNP may be detected in this fashion with greaterprecision relative to EF alone. In the nanopump array configurationillustrated in FIG. 11, dehybridization would be expected to occur asthe circuit is closed between the individual donor reservoirs and thecommon receiving reservoir. Electronic circuitry would be designed sothat a spectrum of voltages, voltage pulses or perhaps cycles ofpolarity reversals, would be applied in sequence between individualdonor reservoirs and the common receiving reservoir. Whendehybridization occurs, the passage of the fluorescent-labeled probe DNAis detected by the optical system. Probe sequence identity is assured byenergizing one donor reservoir at a time. Conditions under which theprobe and capture DNA sequences dehybridize (voltage, and perhapstemperature and ionic strength) would be recorded, correlated with theprobe sequence identity and related to the degree of mismatches basepairs. The flow-through configuration inherent in the nanopump designmay also permit temperature adjustments and ionic gradients to controlde-hybridization.

[0100] The combined effect of electrophoretic force and electroosmoticfluid flow provided by the nanopump design may improves the resolutionof detachment of target probe DNA sequences from capture sequences (FIG.12). Such a system may resolve either detachment (dehybridization) ofprobes containing mismatched base pairs (i.e., base-pair mismatchanalysis) and/or different DNA chain lengths with same degree ofhybridization.

[0101] The flow through feature of the nanopump array also obviates theneed for reversing polarity to drive away non-specific analytes ornonreacted molecules. Buffer is simply flowed across bound probe layerto wash.

[0102] The flow through feature also permits changing the temperatureand ionic strength of the running buffer up-stream of the nanopumparray. This approach may permit the use of temperature gradients (inrange of melting temperature, T_(m) of duplex DNA) and/or ionicgradients in addition to electrophoretic forces and electroosmotic flowto refine dehydridization conditions and thus improve resolution ofmismatched base pairs.

[0103] Although the invention has been described with respect toparticular embodiments and examples, it will be appreciated that avariety of modifications and changes may be made without departing fromthe claimed invention.

What is claimed:
 1. A nanopump comprising a supply reservoir for holdinga supply electrolyte solution, disposed against a wall region of thereservoir, a membrane having a plurality of flow-through channelsextending between an inner membrane surface adapted for contact withsaid supply electrolyte solution, and an outer side adapted for contactwith a recipient electrolyte solution contained outside of the supplyreservoir, where said channels have a minimum cross-sectional dimensionbetween 2 and 100 nm and a net surface charge when the pH of the supplysolution is within a given pH range, electrodes disposed on either sideof membrane for contact with said supply and recipient solutions, and acontroller including a power source operatively connected to saidelectrodes for applying a selected voltage potential across saidchannel, to pump solution in the supply reservoir through said channel.2. The pump of claim 1, wherein said channels have a substantiallyuniform minimum dimension along their lengths.
 3. The pump of claim 2,wherein said channels have a minimum dimension in a selected rangebetween about 2 and 30 nm.
 4. The pump of claim 1, wherein saidcontroller is adapted to apply a voltage potential across the electrodesof between about 0.5-20 volts.
 5. The pump of claim 4, wherein saidchannels have a minimum dimension in a selected range between about 2and 30, and application of a voltage potential across said electrodes ofbetween about 0.5-20 volts is effective to produce a flow rate acrosssaid membrane of between about 0.25 and 1.25 ul/min.
 6. The pump ofclaim 4, wherein said controller includes a control element for applyingacross the electrodes a pulsed voltage whose duration is effective topump a selected volume of supply solution across said membrane.
 7. Thedevice of claim 1, wherein said membrane is formed of a siliconsubstrate with polysilicon and silicon dioxide layers.
 8. The pump ofclaim 1, which further includes a recipient reservoir designed forholding the recipient solution in contact with the outer side of saidmembrane.
 9. A method of pumping controlled and reproducible nanoliterquantities of an electrolyte solution from a supply reservoir into arecipient electrolyte solution comprising placing the electrolytesolution in a supply reservoir, in contact with a membrane having aplurality of flow-through channels extending between an inner membranesurface adapted for contact with said supply electrolyte solution, andan outer membrane surface, said channels having a minimumcross-sectional dimension between 2 and 100 nm and a net surface chargeat the pH of the supply solution, placing the outer membrane surface incontact with a recipient electrolyte solution, and with a pair ofelectrodes placed across said membrane, in contact with solution in saidsupply reservoir and with said recipient electrolyte, applying acrosssaid electrodes, a voltage potential effective to pump supply solutionacross said membrane from the supply reservoir into the recipientsolution.
 10. The method of claim 9, wherein said channels have asubstantially uniform minimum dimension along their lengths, and aminimum dimension in a selected range between about 2 and 30 nm.
 11. Themethod of claim 10, wherein the voltage potential applied across themembrane is between about 0.5 and 20 volts.
 12. The method of claim 9,wherein said recipient electrolyte solution is contained in a recipientreservoir, and said membrane is disposed between the supply andrecipient reservoirs.
 13. A separation device for separatingmacromolecules in a solution of macromolecules having differentmolecular sizes, comprising a separation chamber having upstream anddownstream ends, one or more membranes disposed in said chamber betweensaid upstream and downstream ends, partitioning said chamber into two ormore chamber regions, respectively, where (i) each membrane has aplurality of flow-through channels extending between adjacent chamberregions, (ii) said channels have a selected minimum cross-sectionaldimension in the range between 2 and 100 nm and a net surface chargewhen exposed to a solution within a given pH range, and (iii) if thedevice contains two or more such membranes, the selected minimumcross-section of the channels in any membrane is greater than that inmembrane immediately adjacent in the downstream direction, upstream anddownstream electrodes disposed in said chamber, for contacting solutionplaced in the chamber and contained in the in the upstream-most anddownstream-most of the chamber regions, respectively, and a controllerincluding a power source operatively connected to said electrodes forapplying a selected voltage potential across said channel, to pumpsolution through each of said membranes, in an upstream-to-downstreamdirection, wherein macromolecules contained in said solution arefiltered at each successive membrane, to concentrate successivelysmaller macromolecules in successively more downstream chamber regions.14. The device of claim 13, wherein said device contains at least twomembranes, and at least two separate chamber regions.
 15. The device ofclaim 14, for use in separating globular proteins having solution radiibetween about 20 and 300 angstroms, wherein said membranes have selectedsizes in the same range.
 16. The device of claim 14, which furtherincludes a fluid outlet communicating with each chamber region, and anelectrode associated with each outlet, and said voltage source isoperably connected to said outlet electrodes, for divertingmacromolecules in a chamber region out of the chamber through saidoutlet.
 17. The device of claim 14, wherein each chamber region hasinlet and outlet ports for circulating liquid through that chamber, toremove separated macromolecules in the chamber region from the chamber,18. A method of separating macromolecules in a solution ofmacromolecules having different molecular sizes, comprising: (a) placingthe electrolyte solution in an upstream chamber region of a chamberhaving upstream and downstream chamber regions separated by a membranehaving a plurality of flow-through channels extending between an innermembrane surface adapted for contact with said solution, and an outermembrane surface in contact with electrolyte solution contained in adownstream reservoir, where (i) said channels have a minimum selectedcross-sectional dimension between 2 and 100 nm which is effective toblock passage through the membrane of at least one of thedifferent-sized macromolecules, and (ii) a net surface charge at the pHof said solution, (b) with a pair of electrodes placed across saidmembrane, in contact with solution in said upstream and downstreamreservoirs, applying across said electrodes, a voltage potentialeffective to pump supply solution across said membrane from the upstreaminto the downstream reservoir, wherein macromolecules in said solutionare separated on the basis of their ability to pass through the channelsin said membrane.
 19. The method of claim 18, wherein said chamberincludes at least two membranes, at least three separate chamberregions, and the selected minimum cross-section of the channels in anymembrane is greater than that in the membrane immediately downstream.20. The method of claim 19, which further includes periodically removingmacromolecules accumulated in each of said chamber regions.
 21. A devicefor determining the lengths of nucleic acid fragments in an electrolytesolution of different-length fragments and having a selected pH,comprising a chamber, a membrane disposed in said chamber and having achannel extending between an upstream chamber region adapted to hold theelectrolyte solution of such different-length fragments, and adownstream chamber region adapted to hold an electrolyte solution, where(i) said channel has a selected minimum cross-sectional dimension in therange between 2 and 15 nm and a net surface charge within a given pHrange that includes the selected solution pH, upstream and downstreamelectrodes disposed in said upstream and downstream chamber regions,respectively, for contacting solution placed in the correspondingchamber regions, a controller including (i) a power source operativelyconnected to said electrodes for applying a selected voltage potentialacross said channel, to move individual nucleic acid fragments containedin the solution through said channel, and (ii) a current detector fordetecting time-dependent current flow across said membrane.
 22. Thedevice of claim 21, wherein said channel is formed by a pair of planarchannels, each having a channel thickness in the 2-20 nm range, arrangedorthogonally with respect to one another, such that the intersection ofthe two channels a forms a rectangular area whose dimensions correspondto the two thicknesses of the intersecting channels.
 23. The device ofclaim 21, wherein said membrane has a plurality of channels, and saidcurrent detector is effective to measure time-dependent currentindependently through each of said channels.
 24. The device of claim 21,wherein said minimum channel width is within the range 2-10 nm.
 25. Thedevice of claim 21, wherein said controller is effective to place acrossthe electrodes, a voltage potential effective to move nucleic acidfragments electrophoretically through said channel.
 26. A method fordetermining the lengths of nucleic acid fragments in an electrolytesolution of different-length fragments and having a selected pH,comprising (a) placing the solution in an upstream chamber region of achamber having upstream and downstream chamber regions separated by amembrane having a channel extending between the two chamber regions,where (i) said channel has a selected minimum cross-sectional dimensionin the range between 2 and 15 nm and a net surface charge within a givenpH range that includes the selected solution pH, (b) with a pair ofelectrodes placed across said membrane, in contact with solution in saidupstream and downstream chamber regions, applying across saidelectrodes, a voltage potential effective to pump solution across saidchannel, wherein individual nucleic acid fragments move through saidchannel, and (c) detecting time-dependent changes in current flowthrough said channel, as a measure of the length of individual nucleicacid fragments moving through said channel.
 27. The method of claim 26,wherein said membrane has a plurality of channels, and said detectingincludes detecting time-dependent changes in current flow separatelythrough each of said channels.
 28. The method of claim 26, wherein saidminimum channel width is within the range 2-10 nm.